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GOT1 Inhibition Primes Pancreatic Cancer for Ferroptosis Through the 2 Autophagic Release of Labile Iron 3 4 Daniel M

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GOT1 Inhibition Primes Pancreatic Cancer for Ferroptosis Through the 2 Autophagic Release of Labile Iron 3 4 Daniel M bioRxiv preprint doi: https://doi.org/10.1101/2020.02.28.970228; this version posted February 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 GOT1 Inhibition Primes Pancreatic Cancer for Ferroptosis through the 2 Autophagic Release of Labile Iron 3 4 Daniel M. Kremer1,2, Barbara S. Nelson3, Lin Lin1, Emily L.Yarosz4, Christopher J. 5 Halbrook1, Samuel A. Kerk3, Peter Sajjakulnukit3, Amy Myers1, Galloway Thurston1, 6 Sean W. Hou1, Eileen S. Carpenter5, Anthony C. Andren1, Zeribe C. Nwosu1, Nicholas 7 Cusmano1, Stephanie Wisner1, Johanna Ramos1, Tina Gao1, Stephen A. Sastra6,7, 8 Carmine F. Palermo6,7, Michael A. Badgley6,7,8, Li Zhang1, John M. Asara9,10, Marina 9 Pasca di Magliano11,12,13, Yatrik M. Shah1,5,11, Howard C. Crawford1,11, Kenneth P. 10 Olive6,7, Costas A. Lyssiotis1,5,11‡ 11 12 1 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA 13 2 Graduate Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109, USA 14 3 Graduate Program in Cancer Biology, University of Michigan, Ann Arbor, MI 48109, USA 15 4 Immunology Graduate Program, University of Michigan, Ann Arbor, MI 48109, USA 16 5 Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, 17 MI 48109, USA 18 6 Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, 19 NY 10032, USA 20 7 Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA 21 8 Department of Pathology, Columbia University Medical Center, New York, NY 10032, USA 22 9 Division of Signal Transduction and Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Boston, MA, 23 USA 02115 24 10 Department of Medicine, Harvard Medical School, Boston, MA, USA 02115 25 11 Rogel Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA 26 12 Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA 27 13 Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA. 28 ‡ Correspondence: [email protected] 29 30 Highlights 31 PDA exhibit varying dependence on GOT1 for in vitro and in vivo growth. 32 Exogenous cystine, glutathione synthesis, and lipid antioxidant fidelity are 33 essential under GOT1 suppression. 34 GOT1 inhibition sensitizes pancreatic cancer cell lines to ferroptosis. 35 GOT1 inhibition represses anabolic metabolism and promotes the release of iron 36 through autophagy. 37 38 Summary 39 Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest solid malignancies, 40 with a 5-year survival rate at ten percent. PDA have unique metabolic adaptations in 41 response to cell-intrinsic and environmental stressors, and identifying new strategies to 42 target these adaptions is an area of active research. We previously described a 43 dependency on a cytosolic aspartate aminotransaminase (GOT1)-dependent pathway 44 for NADPH generation. Here, we sought to identify metabolic dependencies induced by 45 GOT1 inhibition that could be exploited to selectively kill PDA. Using pharmacological 46 methods, we identified cysteine, glutathione, and lipid antioxidant function as metabolic 47 vulnerabilities following GOT1 withdrawal. Targeting any of these pathways was 48 synthetic lethal in GOT1 knockdown cells and triggered ferroptosis, an oxidative, non- 49 apoptotic, iron-dependent form of cell death. Mechanistically, GOT1 inhibition promoted 50 the activation of autophagy in response to metabolic stress. This enhanced the 51 availability of labile iron through ferritinophagy, the autolysosome-mediated degradation 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.28.970228; this version posted February 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 of ferritin. In sum, our study identifies a novel biochemical connection between GOT1, 53 iron regulation, and ferroptosis, and suggests the rewired malate-aspartate shuttle plays 54 a role in protecting PDA from severe oxidative challenge. 55 56 Keywords: Pancreatic ductal adenocarcinoma, GOT1, cysteine, glutathione, GPX4, 57 NADPH, redox, reactive oxygen species, ferroptosis, labile iron, autophagy, 58 ferritinophagy, metabolic dependencies 59 60 Introduction 61 Pancreatic ductal adenocarcinoma (PDA) is a notoriously lethal disease. This stems 62 from late-stage diagnosis and the lack of effective therapies1. A defining feature of PDA 63 is the extensive fibroinflammatory reaction that not only regulates cancer initiation, 64 progression, and maintenance, but also promotes its therapeutic resilience2,3,4. Vascular 65 collapse, impaired perfusion, and hypoxia accompany this desmoplastic reaction to 66 produce a nutrient-deprived and harsh tumor microenvironment3,5. PDA cells reprogram 67 their nutrient acquisition and metabolism to support survival and growth under these 68 conditions6,7. 69 70 Our previous work demonstrated that PDA rewire the malate-aspartate shuttle to 71 generate reduced nicotinamide adenine dinucleotide phosphate (NADPH), a major 72 currency for biosynthesis and redox balance (Figure 1A)8. The malate-aspartate shuttle 73 canonically functions to transfer reducing equivalents in the form of NADH from the 74 cytosol into the mitochondria to facilitate oxidative phosphorylation (OxPHOS). In PDA, 75 we found the mitochondrial aspartate aminotransaminase (GOT2) is the primary 76 anaplerotic source for alpha-ketoglutarate (αKG) and generates aspartate. Aspartate is 77 then transferred to the cytosol and transaminated to produce oxaloacetate (OAA) by the 78 cytosolic aspartate aminotransaminase (GOT1). OAA is reduced to malate by cytosolic 79 Malate Dehydrogenase (MDH1) and is then oxidized by Malic Enzyme 1 (ME1) to 80 generate NADPH, which is utilized to support redox balance and proliferation in PDA8. 81 Furthermore, we demonstrated that this non-canonical pathway was orchestrated by 82 mutant KRAS, the signature oncogenic driver of PDA. Specifically, mutant Kras led to 83 the transcriptional upregulation of GOT1 and the concurrent repression of GLUD1, 84 which drives an alternative metabolic pathway. Indeed, a higher expression of GOT1 to 85 GLUD1 is associated with worse patient outcome9. Thus, in an effort to target this 86 rewired metabolic pathway, we have placed our focus on GOT1. And, notably, we and 87 others recently identified drug scaffolds that may serve as leads in the development of a 88 clinical grade GOT1 inhibitor 10,11,12,13. 89 90 Herein, we present a detailed analysis of GOT1 dependence across a large panel of 91 PDA cell lines and specimens. We demonstrate that GOT1 sensitivity varies among the 92 cultures in this panel, is dispensable in non-transformed human lines, and that GOT1 93 inhibition stunted growth in tumor xenograft models. In GOT1 dependent contexts, 94 GOT1 inhibition blocked progression through the cell cycle, leading to cytostasis. Thus, 95 we then sought to characterize metabolic dependencies following GOT1 withdrawal that 96 could be exploited to selectively kill PDA14,15. Examination of a targeted library of 97 metabolic inhibitors in GOT1 knockdown cells led to the discovery that that exogenous 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.28.970228; this version posted February 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 98 cystine was essential for viability following chronic GOT1 suppression. Cystine is used 99 for reduced glutathione (GSH) biosynthesis, which mediates protection against lipid 100 oxidation. GOT1 knockdown in combination with inhibitors of glutathione synthesis or 101 lipid antioxidant machinery led to cell death. We characterized this as ferroptosis: an 102 oxidative, non-apoptotic, and iron-dependent form of cell death. We then determined 103 that GOT1 withdrawal promoted a catabolic cell state that resulted in decreased 104 OxPHOS and the activation of autophagy and ferritinophagy16. Ferritinophagy primed 105 GOT1 knockdown cells for ferroptosis by increasing labile iron pools. Together with the 106 modulation of NADPH and GSH levels, our study demonstrates that GOT1 inhibition 107 promotes ferroptosis sensitivity by promoting labile iron and illustrates how the rewired 108 malate-aspartate shuttle participates in the maintenance of both antioxidant and 109 energetic balance. 110 111 Results 112 GOT1 dependent PDAs require GOT1 for cell cycle progression and proliferation 113 To examine GOT1 dependence in a large panel of PDA cell lines and primary 114 specimens with temporal control, we developed doxycycline (dox)-inducible short 115 hairpin (sh)RNA reagents (iDox-sh) that target the coding and 3’UTR regions of GOT1 116 (sh1 and sh3), or scramble (shNT). shRNA activity was examined phenotypically by 117 assessing colony formation and protein levels following dox treatment (Figures 1B,C 118 and S1A,B). We also measured aspartate levels as a biochemical readout
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